Structure-based ligand discovery for the Large-neutral Amino Acid Transporter 1, LAT-1 Ethan G. Geiera,1, Avner Schlessingera,b,1,2, Hao Fana,b,c, Jonathan E. Gablec,d, John J. Irwina,b,c, Andrej Salia,b,c,3, and Kathleen M. Giacominia,3 Departments of aBioengineering and Therapeutic Sciences and cPharmaceutical Chemistry, bCalifornia Institute for Quantitative Biosciences, and dGraduate Group in Biophysics, University of California, San Francisco, CA 94158 Edited by John Kuriyan, University of California, Berkeley, CA, and approved February 19, 2013 (received for review October 17, 2012) The Large-neutral Amino Acid Transporter 1 (LAT-1)—a sodium- determined by X-ray crystallography (15–19). Structures of the ar- independent exchanger of amino acids, thyroid hormones, and pre- ginine:agmatine antiporter AdiC from Escherichia coli (15, 17, 18) scription drugs—is highly expressed in the blood–brain barrier and and Salmonella enterica (20) in different conformations reveal an various types of cancer. LAT-1 plays an important role in cancer internal twofold pseudosymmetry, similar to the structures of the development as well as in mediating drug and nutrient delivery sodium- and chloride-dependent leucine transporter, LeuT (19, 21). across the blood–brain barrier, making it a key drug target. Here, These data, combined with structures of additional related trans- we identify four LAT-1 ligands, including one chemically novel sub- porters (22) and molecular dynamics (MD) simulations (23), sug- strate, by comparative modeling, virtual screening, and experimen- gest a common transport mechanism among the LAT-1 homologs tal validation. These results may rationalize the enhanced brain and LeuT, in which the role of sodium in LeuT is proposed to be permeability of two drugs, including the anticancer agent acivicin. mimicked by a proton in some APC transporters (23). Thus, LAT-1 Finally, two of our hits inhibited proliferation of a cancer cell line by probably also transports ligands across the cell membrane via the distinct mechanisms, providing useful chemical tools to characterize alternating access transport mechanism (22, 24, 25). the role of LAT-1 in cancer metabolism. In this study, we take an integrated computational and experi- mental approach to characterize previously unknown LAT-1 membrane transporter | polypharmacology | glioblastoma multiforme | ligands. We construct structural models of LAT-1 based on solute carrier (SLC) transporter structures of homologous APC family transporters from pro- karyotic organisms and then perform virtual ligand screening of ( metabolite and prescription drug libraries against these models to arge-neutral Amino Acid Transporter 1 LAT-1) is a sodium- predict small-molecule ligands. The top-scoring hits are tested Lindependent exchanger found in the brain, testis, and placenta, experimentally for LAT-1 inhibition and transport by using cis- where it mediates transport of large-neutral amino acids (e.g., inhibition experiments and trans-stimulation assays, respectively. tyrosine) and thyroid hormones (e.g., triiodothyronine) across the Furthermore, we characterize the effect of select validated ligands cell membrane (1). More specifically, LAT-1 is highly expressed in – on cell proliferation. Finally, we describe the pharmacological the blood- and brain-facing membranes of the blood brain barrier implications of our results, including how the intended and un- (BBB) to supply the central nervous system (CNS) with essential intended effects of the discovered ligands may be mediated by nutrients and to help maintain the neural microenvironment (2). LAT-1 transport across the BBB as well as their potential use as LAT-1 is also an important drug target because it transports chemical tools to characterize the role of LAT-1 in cancer. several prescription drugs, such as the antiparkinsonian drug L-dopa and the anticonvulsant gabapentin, across the BBB, thereby en- Results abling their pharmacologic effects (3, 4). This function at the LAT-1 Predicted Structure and Ligand Binding. LAT-1 was modeled BBB has made LAT-1 a target for drug delivery by modifying based on the X-ray structure of the arginine/agmatine transporter CNS-impermeable drugs such that they become LAT-1 substrates AdiC from E. coli in the outward-occluded arginine-bound con- and have enhanced BBB penetration (5, 6). formation (17) and the structure of the APC transporter ApcT In addition, LAT-1 expression levels are increased in many from Methanococcus jannaschii in an inward-apo conformation types of cancer, including non-small-cell lung cancer and glio- (16) (Fig. S1 and SI Materials and Methods). The final LAT-1 blastoma multiforme (GBM) (7, 8). LAT-1 expression increases model contains the whole transmembrane domain of the protein as cancers progress, leading to higher expression levels in high- (i.e., the 12 transmembrane helices), including the residues con- grade tumors and metastases (9). In particular, LAT-1 plays a key stituting the predicted ligand-binding site. Comparative models role in cancer-associated reprogrammed metabolic networks by were first scored by using Z-DOPE, a normalized atomic distance- supplying growing tumor cells with essential amino acids that are dependent statistical potential based on known protein structures used as nutrients to build biomass and signaling molecules to (26). The Z-DOPE scores of the top models were −0.3, suggesting enhance proliferation by activating progrowth pathways such as that 60% of its Cα atoms are within 3.5 Å of their correct positions the mammalian target of rapamycin (mTOR) pathway (10). Furthermore, inhibiting LAT-1 function reduces tumor cell pro- liferation, indicating that it may be a viable target for novel anti- – Author contributions: E.G.G., A. Schlessinger, J.J.I., A. Sali, and K.M.G. designed research; cancer therapies (11 13). A cancer drug targeting LAT-1 can E.G.G., A. Schlessinger, and J.E.G. performed research; E.G.G., A. Schlessinger, A. Sali, and therefore be a LAT-1 inhibitor that deprives the cancer cells of K.M.G. contributed new reagents/analytic tools; E.G.G., A. Schlessinger, H.F., J.E.G., A. Sali, nutrients or a cytotoxic LAT-1 substrate with an intracellular and K.M.G. analyzed data; and E.G.G., A. Schlessinger, A. Sali, and K.M.G. wrote the paper. target (e.g., a metabolic enzyme). The authors declare no conflict of interest. LAT-1 is a large protein with 12 putative membrane-spanning This article is a PNAS Direct Submission. helices (14). To transport solutes across the membrane, LAT-1 1E.G.G. and A. Schlessinger contributed equally to this work. binds SLC3A2, a glycoprotein with a single membrane-spanning 2Present address: Department of Pharmacology and Systems Therapeutics, Tisch Cancer helix that serves as a chaperone for LAT-1 (14). The atomic Center, Mount Sinai School of Medicine, New York, NY 10029. structure of human LAT-1 is not known, but LAT-1 exhibits sig- 3To whom correspondence may be addressed. E-mail: [email protected] or kathy.giacomini@ nificant sequence similarity to prokaryotic transporters such as ucsf.edu. members of the amino acid/polyamine/organocation transporter This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (APC) family, whose representative structures have been recently 1073/pnas.1218165110/-/DCSupplemental. 5480–5485 | PNAS | April 2, 2013 | vol. 110 | no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1218165110 Downloaded by guest on October 2, 2021 Some of the top-scoring hits were shown previously to be LAT-1 ligands, increasing our confidence in the binding site model. For example, the known substrate L-Trp was ranked 50th in the docking screen of KEGG LIGAND COMPOUND. The 200 (3.1%) KEGG DRUG and 500 (3.9%) KEGG COMPOUND top-scoring hits against our top two models were analyzed manually. A compound was selected for experimental testing based on three criteria: (i) similarity between its docking pose and those of known ligands in complex with LAT-1 (28); (ii) the chemical novelty of its scaffold, especially if it occurred fre- quently among the top-scoring compounds; and (iii) its phar- macological effect (28). Experimental Validation of Predicted Ligands. ALAT-1–overex- pressing cell line was generated by stably transfecting HEK cells with human LAT-1 cDNA. HEK-LAT1 cells expressed 20-fold Fig. 1. Predicted LAT-1 structure and ligand-binding mode. (A)Predicted higher levels of LAT-1 mRNA relative to HEK-EV cells and structure of the LAT-1–phenylalanine complex. LAT-1 (gray) and phenylalanine demonstrated LAT-1–specific uptake of the established system L (cyan) are shown as the stick models; oxygen, nitrogen, and hydrogen atoms substrates, gabapentin and L-leucine (Fig. S3 A–D). Twelve of the are depicted in red, blue, and white, respectively; key hydrogen bonds between top-scoring molecules were selected for experimental testing by phenylalanine and LAT-1 (involving residues Thr-62, Ile-63, Ile-64, Ser-66, Gly-67, cis-inhibition assay (Table 1, Table S2, and Fig. 2). Each molecule Phe-252, Ala-253, and Gly-255) are shown as dotted gray lines. (B) Structures was tested as a LAT-1 ligand by determining its ability to inhibit of representative LAT-1 substrates. Known LAT-1 substrates, including me- transport of a known LAT-1 substrate in HEK-LAT1 cells at tabolites (tryptophan, methionine, and thyroxine) and prescription drugs μ (melphalan, L-dopa, and gabapentin) are shown using MarvinView 5.4.1.1 concentrations of 10 and 100 M (Fig. 3 and Fig. S3E). The known (Chemaxon). LAT-1 inhibitor 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) was also included as a positive control. At 100 μM, in- hibition of intracellular gabapentin accumulation ranged from (27) (Table S1). Each model was also evaluated based on its ability 88% (3,5-diiodo-L-tyrosine) to <10% (cystine, mebendazole, and to discriminate between known ligands and likely nonbinders nocadezole), with the metabolites 3,5-diiodo-L-tyrosine and (decoys), by using enrichment curves derived from ligand-docking 3-iodo-L-tyrosine, as well as the tryptophan hydroxylase inhibitor fi fi calculations (28).